Recent studies have focused on the significance of stress proteins such as heat shock protein (HSP) 70 and intracellular antioxidants. These are considered as an integral part of the cellular defense system and may play a major role in myocardial preservation during ischemic arrest. Unfortunately, a successful molecular model is not available to prove or disprove this hypothesis. The applicatant proposes to develop a transgenic and knockout mouse model for stress proteins HSP 70 and HSP 32, as well as for antioxidant regulating enzymes superoxide dismutase, catalase, and glutathione peroxidase. Myocardial preservation will be examined using isolated perfused working mouse hearts subjected to 30 min of ischemia, followed by 60 min of reperfusion. Matched nontransgenic mice will be used for control experiments. Ischemic reperfusion injury will be assessed by carefully monitoring left ventricular functions and myocardial tissue injury. The levels and location of overexpressed HSPs and antioxidants in the transgenic mice will be studied by Western blot analysis, enzymatic assay and immunohistochemistry. Four major factors contributing to ischemic reperfusion injury--breakdown of high energy phosphate compounds, cellular Ca2+ overloading, free radical generation, sarcolemmal phospholipid loss--in conjunction with the accumulation of lysophosphoglycerides and free fatty acids will be evaluated in an attempt to delineate the mechanism of protection afforded by the transgenic mice. The levels (Western blot), as well as the induction of mRNAs (Northern blot) of the intracellular antioxidants and heat shock proteins, will be examined in the control, transgenic and knockout mice in order to study the interrelationship between the HSPs and antioxidants. If HSPs and antioxidants indeed constitute cellular defense against ischemic reperfusion injury, the transgenic overexpressed mice should be tolerant to injury while the knockout mice will be more susceptible to such injury. The results of these studies will conclusively demonstrate whether constitutive cellular protection against ischemia is provided by intracellular antioxidants and/or HSPs and, if so, the underlying mechanism of this protection.